Aqueous β2&#39;-glycoprotein I composition

ABSTRACT

A method for preparing an aqueous protein composition from human blood plasma, wherein the plasma undergoes a series of treatments by heating and precipitating agent to give albumin solutions containing the precipitating agent, and said precipitating agent is separated from the resulting solutions to give a crude aqueous albumin solution which is subjected to at least one liquid phase chromatography step to retain at least part of the secondary proteins other than albumin. The chromatography comprises affinity chromatography on a particulate support consisting of neutral particles loaded with at least one compound comprising sulphate groups, whereby, after the albumin solution has been fed through, the particulate affinity chromatography support is eluted by feeding through an aqueous saline solution, preferably by increasing the ionic strength, and the desired protein composition is collected by elution. A protein composition obtained by the method, an isolated glycoprotein having a molecular weight of 50,000±3,000 daltons and contained in the composition, an agent for stabilizing albumin during a Tyndall effect treatment, and an agent for detecting and/or assaying antibodies by ELISA or immuno-imprinting, are also provided.

This application is a 35 U.S.C. 371 national stage filing ofinternational application PCT/FR94/00143, filed Feb. 2, 1994.

The present invention relates to a protein composition obtained as asecondary product in the production of albumin from human blood plasma,to a method for producing it, to the glycoprotein which it contains andto the use of the said glycoprotein in albumin-stabilizing agents and asan agent for allowing antibodies to be detected and/or assayed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the turbidity of the albumin solutions Aand B as a function of time.

FIG. 2 is a graph that shows as the protein concentrations increases,the turbidity of albumin decreases.

In the state of the art, it is known that, in order to obtain solutionsof human albumin, blood plasma is fractionated either by the Cohn method(see Cohn J. et al., J. Amer. Chem. Soc, 72, 465-474-(1950)), or by aderived method in which successive thermal and ethanolic treatments arecarried out or a similar method in which a precipitation agent otherthan ethanol (especially ether, ammonium sulfate, polyethylene glycol orcaprylic acid) is used. According to the Cohn method, which is, inpractice, the one used most commonly, the plasma is cooled to -30° C.and then warmed to -2° C., which results in a cryoprecipitate containinganti-hemophilia factor VIII, fibrinogen and fibronectin. Thesupernatant, referred to as "supernatant I", is separated from theabovementioned precipitate; its pH is lowered to 5.85±0.05 and ethanolis added until the ethanolic concentration is 19% by volume, thetemperature being lowered as the ethanol is added, to -5° C. Aprecipitate is thus obtained, referred to as "precipitate (II+III)",containing in particular the gamma-globulins and a supernatant, referredto as "supernatant (II+III)" containing the albumin and impurities. Thesupernatant thus obtained is taken up and the alcohol content isincreased until an ethanolic concentration of 40% by volume is obtained,the temperature being lowered to about -8° C. A precipitate is thusobtained, referred to as "precipitate IV", and a supernatant, referredto as "supernatant IV", which contains the albumin in a degree of purityof about 94 to 97% by weight. The supernatant IV serves as a startingmaterial for the desired albumin solutions, but it contains a largeamount of ethanol; according to a first technique, the supernatant IVmay be dialyzed directly against physiological saline, but a very largeamount of water then needs to be used and the method is thus slow andexpensive; according to another technique, the pH of the supernatant IVis lowered to 4.80±0.05, thereby causing the albumin to precipitate out:this precipitate, referred to as "precipitate V", is separated out andis redissolved in physiological saline, the rest of the ethanol beingextracted by dialysis.

The aqueous solutions obtained after dialysis of the "supernatant IV" orof the "precipitate V" which is redissolved contain albumin and about 4%by weight, relative to the total weight of protein material, of otherso-called secondary or contaminating proteins and/or polymers.

According to FR-A-2,690,444, the albumin is separated from the otherproteins by a liquid phase chromatographic method in which, afterdialysis, the aqueous solution containing the albumin is passed throughat least one so-called "hydrophobic" chromatography column filled with aparticulate material capable of retaining some of the proteins otherthan the albumin; in order to complete the separation, it is alsoproposed to pass the aqueous albumin solution through at least oneaffinity chromatography column containing a neutral particulate supportor a particulate support close to neutrality charged with a polysulfatedcompound. The effluent obtained by this method consists of a solution ofpurified albumin, the majority of the secondary proteins being boundeither to the hydrophobic chromatography column(s) or to the affinitychromatography column(s).

According to a preferred embodiment of this method, an aqueous albuminsolution at a pH between 6.5 and 7.8 and at a concentration of greaterthan 1 g/l and less than 400 g/l is subjected to the chromatography.

A neutral particulate support whose particles are charged with acompound containing. hydrophobic C₃ -C₈ alkyl radicals is advantageouslychosen as particulate material capable of retaining the contaminatingproteins in the hydrophobic chromatography; a compound containing butylradicals may in particular be chosen as compound charging the particlesof the support: the packing bed consists, for example, of the productmarketed by the company "Merck" under the trade name "FRACTOGEL TSKBUTYL-650". The affinity chromatography is preferably performed ondextran sulfate gel. In order to carry out the affinity chromatographyand the hydrophobic chromatography, a particulate material having a meanparticle size of less than 300 μm is preferably chosen. Thechromatography flow rate is generally between 10 and 20 cm/hour and thetemperature between 2 and 25° C. The affinity chromatography may becarried out after or, preferably, before the hydrophobic chromatography.

According to the present invention, it has been observed that thesecondary proteins bound to the packing beds of the abovementionedchromatography columns could be eluted and useful protein compositions,useful especially in the pharmaceutical field, could thus be obtained.

The first subject of the present invention is consequently a method forobtaining an aqueous protein composition from human blood plasma, inwhich the plasma is subjected to successive treatments by the action oftemperature and by the action of a precipitation agent in order to lead,after at least partial separation of the factors VIII and IX and of thegamma globulins, to albumin solutions containing the precipitationagent, followed by separation, especially by dialysis, of theprecipitation agent from the solutions obtained in order to obtain acrude aqueous albumin solution, and the said crude aqueous albuminsolution is subjected to at least one liquid phase chromatography inorder to retain at least some of the secondary proteins other than thealbumin, the said liquid phase chromatography including an affinitychromatography on a particulate support consisting of charged particlesof at least one compound containing sulfate groups, characterized inthat, after passage of the albumin solution, the particulate support ofthe abovementioned affinity chromatography lacuna! is subjected to anelution and in that the desired protein composition is collected onelution.

The elution is preferably carried out by increasing the ionic strengthby passage of a saline solution. The saline solution is preferably anNaCl solution, at a concentration at least equal to 0.3M, advantageouslyof 2M; it is advantageously preceded by a washing operation. Theaffinity chromatography support is washed in particular using a salinebuffer, of molarity equal to 0.16 mol/l, preferably a phosphate bufferconsisting of mono- and disodium phosphates, in particular at aconcentration of 0.01 mol/l, and of sodium chloride, in particular at aconcentration of 0.15 mol/l, in proportions giving a pH of 7.00±0.05.The washing operation is continued for as long as the optical density ofthe effluent is greater than a predetermined value, for example 0.1 ODU(optical density unit).

The solution obtained by eluting the affinity chromatography column(s)contains a mixture of proteins. Another subject of the present inventionis the protein composition thus obtained. This composition contains aglycoprotein which has a molecular weight of 50,000±3000 dalton after orwithout reduction by 2-mercaptoethanol. The glycoprotein constitutesfrom 5 to 100% by weight of the total protein content of the compositionobtained according to the mode of preparation; the elution generallymakes it possible to obtain a weight concentration between 0.05 g and 30g of glycoprotein per liter of eluate, but this concentration may bereduced by dilution or increased by concentration of the eluate.

The glycoprotein isolated according to the invention has thecharacteristics and properties given below.

The first 20 amino acids of its N-terminal region have been determined,in particular by gas phase microsequencing using an "Applied BiosystemsInc, model 470" machine coupled to a phenylrheohydantoin analyzer model120 A (ABI)(SEQ ID NO:1)Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp-Asp-Leu-Pro-Phe-Ser-Thr-Val-Val-Pro-Leu-Lys-Thr.This sequence of the first 20 amino acids corresponds to that of aplasma β2-glycoprotein described (1) in the article by J. Lozier et al.Proc. Natl. Acad. Sci. ISA Vol. 81, pages 3640-3644, June 1984 and (2)in the article by T. Kristensen et al. FEBS Letters, Vol. 289, 1991,pages 183-186.

The amino acid composition of the glycoprotein isolated according to theinvention was determined on two different preparations by two differentlaboratories:

a) the first preparation A was treated by acidic hydrolysis in 5.7M HClfor 24 hours at 110° C. with a "BECKMAN 6300" amino acid analyzer linkedto a "GOLD" system for interpretation,

b) the second preparation B was also analyzed with a "BECKMAN 6300"amino acid analyzer after hydrolysis by 6N HCl for 24 hours and 72hours.

The results obtained are given in Table I below, where they are comparedwith the results published by J. Lozier et al. (1) and T. Kristensen etal. (2). These results show that the glycoprotein is a β2'-glycoproteinI similar to the β2-glycoprotein I described by J. Lozier et al. and T.Kristensen et al.

However, this glycoprotein differs from β2-glycoprotein I. Indeed, afterpurification, precipitation of the glycoprotein according to theinvention with acetone and dialysis against distilled water, sequencingresults in the identification of 14 amino acids, emerging in pairs, andcorresponding to the N-terminal region of the protein. Given the degreeof purity of the protein preparation, this corresponds to cleavage ofthe starting protein. Indeed, 7 of these amino acids correspond to theN-terminal region. The sequence concerned is as follows (SEQ ID NO:2):

Gly-Arg-Thr-Cys-Pro-Lys-Pro The other 7 amino acid, by deduction,correspond to the peptide (SEQ ID NO:3):

Phe-Trp-Lys-Ser-Asp-Ala-Ser This peptide corresponds to the regionbetween amino acids 315 and 321 which has been described by J. Lozierfor β2-glycoprotein I but with a difference of one amino acid. Thisinvolves a serine in place of a threonine. The glycoprotein isolatedaccording to the invention is thus a β2-glycoprotein I allotype, whichwill be referred to hereinbelow as β2'-glycoprotein I.

After cleavage of the protein with cyanogen bromide, separation of thepeptides obtained and analysis of the first three amino acids in eachpeptide, the results described by J. Lozier et al. are found.

A second subject of the present invention is thus the proteincomposition obtained by elution of the affinity chromatography column(s)in the method described above and the β2'-glycoprotein I, as definedabove.

                                      TABLE I    __________________________________________________________________________    GLYCOPROTEIN isolated    Preparation A                Preparation B   β2-GLYCOPROTEIN I    Hydrolysis 24 H                Hydrolysis 24 H                        Hydrolysis 72 H                                According to (1)                                        According to (2)        %          %       %    %       Number of                                               %    Amino        of number  of number                           of number                                of number                                        residues/moles                                               of number    acids        of residues                nmol                   of residues                        nmol                           of residues                                of residues                                        of protein                                               of residues    __________________________________________________________________________    Asp 9.68    6.637                   8.961                        6.092                           9.071                                8.9     28     8.723    Thr 7.89    6.000                   8.101                        5.574                           8.299                                8.3     27     8.411    Ser 5.01    4.609                   6.223                        3.827                           5.698                                5.8     20     6.231    Glu 8.37    6.130                   8.276                        5.340                           7.951                                7.3     24     7.477    Gly 7.62    5.550                   7.493                        5.382                           8.014                                7.1     23     7.165    Ala 5.91    4.490                   6.062                        3.911                           5.823                                5.2     17     5.296    Cys 1.59    3.640                   4.914                        3.497                           5.207                                6.7     22     6.854    Val 5.87    3.180                   4.293                        3.564                           5.307                                5.5     17     5.296    Met 1.08    0.757                   1.022                        0.826                           1.230                                1.2      4     1.246    Ile 3.95    2.275                   3.072                        2.618                           3.898                                4.0     13     4.050    Leu 5.78    4.405                   5.947                        3.841                           5.719                                5.2     18     5.607    Tyr 4.38    3.440                   4.644                        2.682                           3.993                                4.3     14     4.361    Phe 5.64    4.167                   5.626                        3.906                           5.816                                5.5     18     5.607    His 1.65    1.350                   1.823                        1.090                           1.623                                1.5      5     1.558    Lys 10.14   6.809                   9.193                        5.880                           8.755                                9.2     30     9.346    Trp                                  5    Arg 3.31    2.258                   3.049                        1.866                           2.778                                3.1     10     3.115    Pro 12.13   8.370                   11.301                        7.265                           10.817                                9.5     31     9.657    __________________________________________________________________________

It was found that the protein composition and the β2'-glycoprotein areantigenic, that is to say that they are detected by the ELISA method orby immuno-imprinting, using human antibodies, for example forindividuals infected with HBV, HIV or Gougerot-Sjogren syndrome; theyare also recognized under certain conditions, in ELISA, by monoclonalantibodies specific for the p 25/p 55 gag of the HIV1 virus (Ac. Mo.RL4.72.1)and for the antioncogenic protein p 53 (Ac. Mo. 122); thecomposition and β2'-glycoprotein I may thus be used for thecharacterization and assay of antibodies against this protein inpatients suffering from AIDS, hepatitis B, leukemias, Gougerot-Sjogrensyndrome or myelomas. The ELISA method is described, for example, in thearticle by Engvall et al. Immunochemistry 1971, 8, pages 871 to 879 andthe immuno-imprinting method is described, for example, by Towbin et al.Proc. Natl. Acad. Sci. USA, 1979, 76, pages 4350-4354.

The protein composition and β2'-glycoprotein I inhibit the denaturationof albumin at 60° C. observed in turbidimetry, in a "dose-dependent"manner; it is thus possible to use it sic! in order to stabilizealbumin, especially during tyndallization.

β2'-Glycoprotein I may be separated from the secondary proteins withwhich it is mixed in the protein composition obtained by elution of theaffinity chromatography column, in the manner described below: thecomposition is eluted from dextran sulfate gel by an NaCl solution atleast equal to 0.3M, advantageously in the region of 2 mol per liter;the eluate obtained is diluted in saline buffer so as to lower its ionicstrength to less than 0.2; the solution obtained is subjected to achromatography on phosphate gel, and the glycoprotein bound to thephosphate gel is then eluted by increasing the ionic strength with asaline buffer preferably having an ionic strength of greater than 0.4.The flow rate of the phosphate gel chromatography is preferably between10 and 20 cm/hour and the temperature is preferably between 0and 40° C.,preferably 2 and 25° C., under usual conditions.

Another subject of the invention is an agent for stabilizing albuminduring tyndallization, containing the protein composition or theβ2'-glycoprotein I defined above and an agent which makes it possible todetect and/or assay antibodies from humans infected with HBV, HIV,Gougerot-Sjogren syndrome or myelomas, by the ELISA method or byimmuno-imprinting, characterized in that it contains the proteincomposition or the β2'-glycoprotein I defined above.

In order to gain a better understanding of the invention, a mode ofpreparation of the β2'-glycoprotein I according to the invention will bedescribed below, by way of example which is purely illustrative andnonlimiting.

The starting material used is a human plasma fractionated according tothe method described by Kistler and Nitschman (Vox Sang. 7, 414-424(1962)), which is a method derived from that of Cohn, the startingfractions containing the albumin being either the supernatant IV or theprecipitate V.

When starting with the supernatant IV, which comprises 40% (by volume)of ethanol and which has a pH of 5.85±0.05, the supernatant is dilutedby half with a 7 g/l solution of NaCl. The pH is adjusted to 7.45±0.05with 1N sodium hydroxide solution. The albumin is preconcentrated to 90g/l in an ultrafiltration cassette of "Omega" type (Filtron) used in a"Minisette SS Cell NPT Cell" ultrafiltration system (Filtron Techn.Corp.). This cassette has a 0.07 m² filtering surface area and aretention threshold of 30 KDa. Circulation is provided by a peristalticpump at a pressure which varies from 2×10⁵ Pa at the start of theoperation to 5×10⁵ Pa towards the end. When an albumin concentration of90 g/l has been reached, a 9 g/l solution of NaCl is added to thealbumin solution and the dialysis is continued at constant volume untilan ethanol content of less than 0.1% by volume is obtained. When theethanol has been thus removed, the dialysis is continued byconcentrating the solution to 200 g of albumin per liter. The pH isadjusted to 7.10±0.05 with 1N hydrochloric acid solution.

When starting with the precipitate V, the said precipitate isresuspended in physiological saline (NaCl solution at a concentration of9 g per liter) using 4 liters of physiological saline per kilogram ofprecipitate. Filtration is carried out in order to remove lumps ofprecipitate which have not been resuspended during stirring; the pH isadjusted to 7.45±0.05 with 1N sodium hydroxide solution and the solutionthus obtained is concentrated to 90 g of proteins per liter using thesame dialysis system as described before for the supernatant IV. Thedialysis is then continued by adding physiological saline and working atconstant volume until a final ethanol content of less than 0.1% byvolume is obtained. When the ethanol has been thus removed, the dialysisis continued in order to concentrate the solution to about 200 g ofproteins per liter. The pH is adjusted to 7.10±0.05 with 1N hydrochloricacid solution.

The albumin solution thus prepared from the supernatant IV or from theprecipitate V then undergoes sterile filtration ("Millex-GS" 0.2 micronfilter (Millipore)).

The crude aqueous albumin solution obtained first undergoes an affinitychromatography. This chromatography is performed in a 50 ml column (2.5cm×10 cm) (Biorad), filled with a particulate material sold by thecompany "Sigma" under the trade name "DEXTRAN BEADS SULFATED". Thecolumn is pre-equilibrated by passing 3 column-volumes of physiologicalsaline through the bed. The albumin solution is then put on this columnand the effluent may be recovered for the production of albumin.

The progress of the chromatography is monitored by measuring the opticaldensity at 280 nm of the fractions leaving the column. The column flowrate is 16 cm/ hour; the chromatography is carried out between 20 and25° C.

After passage of the albumin recovered as effluent, the affinity columnis washed with a saline buffer, close to isotonicity, preferably abuffer consisting of mono- and disodium phosphates, in particular at aconcentration of 0.01 mol/l, and of sodium chloride, in particular at aconcentration of 0.15 mol/l, having a pH of 7.00±0.05, until the opticaldensity of the effluent is less than 0.1. The proteins bound to theaffinity column are then eluted, by increasing the ionic strength, bypassage of 2M NaCl solution. The washing operation and the elution arecarried out at room temperature, the washing and elution flow ratesbeing 16 cm/h.

SDS-Page denaturing electrophoresis was carried out on the solutionobtained. The presence was observed of a diffuse band corresponding tothe presence of a protein having a molecular weight of 50,000±3000daltons after or without reduction by 2-mercaptoethanol. The Schiffcoloration technique shows that this protein is a glycoprotein. Aftercoloration of the gel with Coomassie blue, in this experiment, study ofthe surface area of the optical density peaks shows that the compositioncontains 55% by weight of β2'-glycoprotein I relative to the totalproteins.

The β2'-glycoprotein I is then separated out and purified in thefollowing way: the protein composition obtained by elution of theaffinity chromatography column(s) using a saline solution of NaCl at aconcentration of 2 mol/liter, is diluted 10 times in a buffer consistingof mono- and disodium phosphates, in particular at a concentration of0.01 mol/liter, in proportions giving a pH of 6.8±0.05. It thenundergoes a chromatography on phosphate gel. This chromatography isperformed in a 50 ml column (2.5 cm×10 cm) (Biorad), charged with aparticulate material sold by the company "Bio-Rad" under the trade name"BIO-GEL HTP". The column is preequilibrated by passing through the bed3 column-volumes of buffer consisting of mono- and disodium phosphates,in particular at a concentration of 0.01 mol/liter, in proportionsgiving a pH of 6.8±0.05. The protein eluate of the above chromatography,once diluted, is then put on this column. The chromatography is carriedout with a flow rate of about 15 cm/hour at a temperature of 20° C. Theeffluent is removed and the chromatographic support is then washed usinga phosphate buffer identical to that which served to equilibrate thecolumn. The washing operation is continued for as long as the opticaldensity of the effluent is greater than a predetermined value, forexample 0.1 ODU (optical density unit).

The β2'-glycoprotein I which, under these conditions, binds to thephosphate gel, is then eluted by increasing the ionic strength. Theelution operation is preferably performed using a KCl solution having aconcentration in the region of 1M. This elution solution consists ofmono- and disodium phosphates, in particular at a concentration of 0.01mol/liter, in proportions giving a pH of 6.8±0.05 and of 1 mol/literKCl. The progress of the chromatography is monitored by measuring theoptical density at 280 nm of the fractions leaving the column. The flowrate for equilibrating, loading, washing and eluting the column is 16cm/h. The chromatography is carried out at 20° C.

The eluate is recovered and then dialyzed, preferably in an isotonicbuffer. By monitoring the progress of the chromatography, it is observedthat at least 0.5 g of β2'-glycoprotein I can be loaded per liter ofchromatography bed.

The eluate obtained from the hydroxyapatite gel (BIO-GEL HTP) wasanalyzed by SDS-Page denaturing electrophoresis. The presence of asingle band was observed, corresponding to the presence of a proteinhaving a molecular weight of 50,000±3000 daltons, after or withoutreduction by 2-mercaptoethanol. The purity of this preparation wastested on so-called "HPLC" chromatography on "FRACTOGEL TSK 3000 SW"(Tosho machine) and the presence of a single protein peak is alsoobserved.

The glycoprotein thus isolated has the sequence and compositioncharacteristics mentioned above in the present description, which showthat this is a β2-glycoprotein I allotype: β2'-glycoprotein I.

The examples given below show the stabilization of albumin by thecomposition containing β2'-glycoprotein I.

EXAMPLE 1

Two albumin solutions, A and B, are prepared, of the same proteinconcentration (90 mg/ml), of the same pH (7.1) and of the same ionicstrength. Solution A is an albumin solution obtained according to theprocess described above but which has not undergone chromatographytreatment, and solution B is the solution of purified albumin obtainedafter affinity chromatography on a dextran sulfate gel in order toremove in particular the protein composition retained on thechromatography column. These two albumin solutions A and B are incubatedat 60° C. Hourly, and for 5 hours during the incubation, the turbidityof each solution is measured using a RATIO XR tubidimeter (HACH 43900).

FIG. 1, of turbidity as a function of time, shows a turbidity, due toheat, which is greater for the albumin solution B than for the albuminsolution A, thereby indicating that an inhibitor of heat-induced proteindenaturation has been removed from the albumin A by means of thechromatographic steps.

EXAMPLE 2

Increasing quantities of eluate from the affinity chromatography ondextran sulfate constituting the protein composition are added to thealbumin solution B described in the above example, which has a proteinconcentration of 70 mg/ml. FIG. 2 shows that when the concentration ofthis eluate increases, the turbidity of the albumin decreases afterheating for 5 hours at 60° C. This curve demonstrates, on the one hand,that the protein composition acts as a tyndallization inhibitor and, onthe other hand, that this inhibition is dose-dependent.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GlyArgThrCysProLysProAspAspLeuProPheSerThrValVal    151015    ProLeuLysThr    20    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GlyArgThrCysProLysPro    15    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    PheTrpLysSerAspAlaSer    15    __________________________________________________________________________

We claim:
 1. A method for obtaining an aqueous glycoprotein compositioncontaining β2'-glycoprotein I from human plasma, said methodcomprisingi) alternately cooling and heating said plasma to obtain afirst precipitate containing antihemophiliac factor VIII, fibrinogen andfibronectin and a supernatant; ii) separating the supernatant from saidfirst precipitate and acidifying the supernatant; iii) adding aprecipitation agent to the supernatant while cooling to obtain anaqueous albumin solution and a second precipitate containinggammaglobulin; iv) separating said aqueous albumin solution from saidsecond precipitate and from said precipitation agent; v) subjecting saidaqueous albumin solution to affinity chromatography on a particulatesupport consisting of charged neutral particles, wherein at least onecompound in said particles contains a sulfate group, so that saidglycoprotein is bound to said particles and albumin remains in solution;and vi) eluting said particulate support to obtain an aqueousglycoprotein composition containing β2'-glycoprotein I.
 2. The methodaccording to claim 1 wherein elution is accomplished by passage of asaline solution of increasing ionic strength over said particulatesupport.
 3. The method according to claim 2 wherein said saline solutionis an NaCl solution.
 4. The method according to claim 2 wherein elutionis preceded by at least one washing operation.
 5. The method accordingto claim 4 wherein the washing operation is carried out using a salinebuffer substantially isotonic to human plasma, in proportions such thatthe pH is 7.00±0.05 during the washing operation.
 6. An aqueousglycoprotein composition obtained by the method of claim
 1. 7. Thecomposition of claim 6 having the property of stabilization of albuminduring its tyndalization.
 8. A composition for the detection or assay ofantibodies from humans suffering from hepatitis B virus, humanimmunodeficiency virus, Gougerot-Sjogren syndrome or myelomas usingELISA or immunoimprinting, comprising the glycoprotein composition ofclaim
 6. 9. β2'-glycoprotein I contained in the aqueous proteincomposition according to claim 1, having a molecular weight of50,000±3000 daltons as determined by SDS gel electrophoresis, after orwithout reduction by 2-mercaptoethanol, in which the first 20 aminoacids of the N-terminal region are as follows:Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp-Asp-Leu-Pro-Phe-Ser-Thr-Val-Val-Pro-Leu-Lys-Thr-(SEQ ID NO:1) and the sequence of amino acids 315 to 321 isPhe-Trp-Lys-Ser-Asp-Ala-Ser (SEQ ID NO:3).
 10. The composition according to claim 6 comprising from 5 to 100% by weight of a glycoproteinrelative to the total protein content said glycoprotein having amolecular weight of 50,000±3000 daltons as determined by SDS gelelectrophoresis after or without reduction by 2-mercaptoethanol, inwhich the first 20 amino acids of the Nterminal region are as follows:Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp-Asp-Leu-Pro-Phe-Ser-Thr-Val-Val-Pro-Leu-Lys-Thr-(SEQ ID NO:1) and the sequence of amino acids 315 to 321 isPhe-Trp-Lys-Ser-Asp-Ala-Ser (SEQ ID NO:3).
 11. The method according toclaim 1 having the further steps ofvii) trea ting the aqueousglycoprotein composition of step vi on dextran sulfate by an NaClsolution with a concentration of at least 0.3M per liter to obtain aneluate; viii) diluting the eluate obtained with a saline buffer so as tolower its ionic strength to at most 0.2; ix) subjecting the eluate tochromatography on phosphate gel; and x) adding a saline buffer of ionicstrength greater than 0.4 to the chromatography solution to increase theionic strength, thereby eluting glycoprotein bound to the phosphate gel.12. The method according to claim 11 wherein the chromatography of stepsix and x has a flow rate between 10 and 20 cm/hour and the temperatureduring chromatography is between 0° C. and 40° C.